Alzheimer's disease (AD) is the most common cause of dementia, affecting an estimated 5.3 million individuals in the US alone. However, the underlying cellular and molecular pathophysiology of AD remains largely unknown. The AD brain is characterized by the presence of amyloid plaques and neurofibrillary tangles. The discovery of amyloid beta (A?) as the main component of these amyloid plaques, combined with the identification of AD-causing familial mutations in the gene encoding amyloid precursor protein (APP) led to the amyloid cascade hypothesis, which suggests that A? is the pathogenic culprit and that reducing A? load can improve or prevent AD symptoms. Growing evidence indicates that the intracellular trafficking routes of APP have a major impact on its ability to be processed by ?- and ?-secretase, in the production of A?. Here, we will address the molecular mechanisms controlling the sorting of APP through the endosomal system since recent work indicates that the endosomal membrane is likely to be a primary locale where APP interacts with its cleavage enzymes, ?- and ?-secretase. Specifically, we have obtained evidence implicating APP ubiquitination and the endosomal complex required for transport (ESCRT) in amyloidogenic processing. ESCRT recognizes ubiquitinated proteins on the limiting endosomal membrane and sorts them into intraluminal vesicles (ILVs) in the interior of multivesicular endosomes. We have identified candidate lysine residues in the APP cytodomain that undergo ubiquitination and control the ability of APP to sort into ILVs. Interestingly, mutating these residues leads to a selective increase in the generation of A?40, the more abundant and less pathogenic species of A?. Although APP ubiquitination appears to be a critical signal in its trafficking, the E3 ubiquitin ligase(s) acting on APP has not been identified. F-box and leucine-rich repeat protein 2 (FBL2) is a promising candidate as part of the Skp1-Cullin-F box (SCF) E3 ligase complex. The specific aims of this proposal will be: (i) To precisely map the ubiquitination sites of APP and determine the type(s) of ubiquitin modification bound to this protein; (ii) To investigate the role of FBL2 in APP ubiquitination at the endosomal membrane; and (iii) To determine the in vivo effects of APP ubiquitination deficiency on A? levels and amyloid plaque generation. We anticipate that the experiments detailed in this proposal will provide a better understanding of mechanisms regulating the trafficking and processing of APP through the endosomal system and identify potential new targets for urgently needed AD therapeutics.